Phospholipids are a diverse group of molecules and are major constituents of animal cell membranes. Although there is a great deal of data concerning the phospholipid makeup of membranes, little is known about the factors which regulate the proportions of phospholipid species found in a particular biological membrane. Specific functional roles for most of the individual phospholipid species have not been described. The long-term goal of the project will be to define the mechanisms involved in regulating the phospholipid composition and the biosynthesis of phospholipids in animal cells. Screening procedures will be developed and used to isolate mutants in cultured animal cells which are defective in various aspects of phospholipid synthesis. The mutants will be used as tools to identify regulatory factors which govern the biosynthesis of phospholipids, to isolate the genes and gene products involved, and to identify roles for specific phospholipid species in cellular processes. Specific objectives during the upcoming grant period are: 1) Methods will be developed and used to isolate Chinese hamster ovary cell mutants whcih are defective in two dihydroxyacetonephosphate acyltransferase (DHAPAT) activities: the peroxisomal and the microsomal forms. These mutants will be characterized and used to identify roles which each activity plays in the synthesis of diacyl phospholipids and in the synthesis of ether-linked phospholipids. The mutants defective in the microsomal DHAPAT activity will be used to determine if this activity and the microsomal glycerol-3- phosphate acyltransferase activity are a function of the same gene product; 2) Mutants have been isolated in a macrophage tumor-cell line, RAW 264.7, which have greatly reduced (80-90%) levels of plasmalogens, and ether-linked lipid. A role for plasmalogens in the responses of the macrophage to immunological stimuli has been proposed previously. This hypothesis will directly addressed by examining the ability of the plasmalogen-deficient mutants to demonstrate responses to stimuli which are characteristic of the wild-type macrophage cell line. These studies will yield better understanding of the regulatory aspects of phospholipid synthesis and the role of a specific class of lipid, ether lipids, in cellular function.